SUMMARY/ABSTRACT Tuberculosis remains a severe global health problem. A better understanding of Mycobacterium tuberculosis, the bacterium responsible for this disease, will facilitate development of new anti-tuberculosis strategies. As in other bacterial pathogens, the secreted and surface-localized proteins of M. tuberculosis have important roles in virulence and in the development of host immunity. The long-term objective of this research is to define the systems responsible for exporting these proteins to their proper location and determine the role they play in M. tuberculosis pathogenesis. Our research has helped show mycobacteria to possess conserved protein export pathways: the general secretion (Sec) pathway and the twin-arginine translocation (Tat) pathway. We also identified a specialized system that is dedicated to a select subset of secreted and surface proteins. This SecA2-dependent export system is important to virulence as shown by the attenuated phenotype of a M. tuberculosis secA2 mutant in macrophages and mice. Macrophages infected with the secA2 mutant produce higher levels of pro-inflammatory cytokines and effectors. We hypothesize that the role of SecA2 in virulence is to limit host immune responses, possibly through modulation of MyD88-dependent pathways. The specific aims of this proposal are the following. 1) Investigate the role of SecA2 in limiting host responses and promoting growth in macrophages by performing a global analysis of macrophage responses altered by SecA2 and testing the significance of the immunomodulation in macrophages and mice. 2) Identify SecA2-dependent proteins by quantitative proteomics and study the relationship between SecA2 and newly identified Dos regulated cell envelope proteins. 3) Characterize the mechanism of SecA2 export by identifying targeting domains in SecA2-exported proteins and by identifying proteins that work with SecA2 in export. The research in this proposal will improve our understanding of the role played by the SecA2 export system in M. tuberculosis pathogenesis and it will clarify the mechanistic basis of this new type of specialized export pathway. The results may reveal new approaches for disease intervention and facilitate construction of live mycobacterial vaccines with enhanced ability to export and present protective antigens.